Figure 5

DTX3L and ARTD9 repress tumor suppressor IRF1 expression in mPCa cells. (A and B) Immunoblot analyses of the tumor suppressor gene product IRF1. PC3-siMock, PC3-siDTX3L (A) or PC3-siARTD9 (B) single knockdown cells were treated with or without IFNγ (200 U/ml) for 6 h and then whole cell extracts separated by SDS PAGE, blotted and subsequently probed with antibodies for ARTD9, DTX3L, IRF1, IRF7 and tubulin. The immunoblots are representative of at least three independent experiments. (C) Quantification of IRF1 levels shown in Figure 5A, B. IRF1 levels were normalized to tubulin. Values represent the means of three independent experiments. (D) Quantification of ARTD9 and DTX3L protein levels in PC3-siMock, PC3-siDTX3L and PC3-siARTD9 single knockdown, respectively, as represented in Figure 5A, B. ARTD9 and DTX3L protein levels were normalized to tubulin. Values represent the means of three independent experiments. (E) Cell proliferation analyses of PC3-CMVprom-empty-control and PC3-CMVprom-IRF1 cells were assessed in presence or absence of IFNγ (100U/ml) by the trypan blue exclusion assay. Relative cell proliferation and cell numbers are presented as means of three independent experiments performed in triplicates. (F) Cell proliferation analyses of PC3-siMock, PC3-siIRF1 and PC3-siARTD9 single knockdown cells were assessed in presence or absence of IFNγ (200 U/ml) by the trypan blue exclusion assay. Relative cell proliferation and cell numbers are presented as means of three independent experiments performed in triplicate. (G) Cell proliferation analyses of LNCaP-siMock and LNCaP-siIRF1 single knockdown cells were assessed by the trypan blue exclusion assay. Relative cell proliferation and cell numbers are presented as means of two independent experiments performed in triplicate. All error bars shown in A to G represent the SE. Statistical analysis was performed using the Student's t test. *P < 0.05, **P < 0.001 and ***P < 0.0001.